KR102368348B1 - Nucleic acid-containing fermented seasoning and manufacturing method thereof - Google Patents
Nucleic acid-containing fermented seasoning and manufacturing method thereof Download PDFInfo
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- KR102368348B1 KR102368348B1 KR1020187034712A KR20187034712A KR102368348B1 KR 102368348 B1 KR102368348 B1 KR 102368348B1 KR 1020187034712 A KR1020187034712 A KR 1020187034712A KR 20187034712 A KR20187034712 A KR 20187034712A KR 102368348 B1 KR102368348 B1 KR 102368348B1
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- South Korea
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- nucleic acid
- moromi
- acid
- fermented seasoning
- producing
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- 239000003643 water by type Substances 0.000 description 1
- 235000015099 wheat brans Nutrition 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/20—Synthetic spices, flavouring agents or condiments
- A23L27/23—Synthetic spices, flavouring agents or condiments containing nucleotides
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/20—Synthetic spices, flavouring agents or condiments
- A23L27/24—Synthetic spices, flavouring agents or condiments prepared by fermentation
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/40—Table salts; Dietetic salt substitutes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/50—Soya sauce
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2300/00—Processes
- A23V2300/14—Extraction
Abstract
5'-뉴클레오티드류를 외부에서 첨가하지 않고, 발효 유래의 5'-뉴클레오티드류만으로 핵산을 고농도로 함유하는 핵산 함유 간장풍 조미료 및 그 제조 방법을 제공하는 것을 목적으로 한다.
대두 또는 밀 등을 주원료로 한 곡물 원료에, 국균을 접종하여 고체 누룩을 조제하고, 물 또는 식염수를 첨가하여 식염 농도 4%(w/v) 이하의 모로미를 가온 분해하는 공정과, 상기 모로미를 가열 처리하여 포스파타아제를 실활시킴으로써 살균 모로미를 조제하는 공정과, 상기 살균 모로미에 핵산 생산 미생물을 접종하여 위해 미생물의 혼입을 억제할 수 있는 용기에서 핵산 발효를 행하는 공정과, 상기 핵산 생산 미생물을 자기 소화하여 균체 내의 RNA를 모로미 중에 유리시킴으로써 RNA 추출물을 조제하는 공정과, 상기 RNA 추출물에 뉴클레아제 및 데아미나아제를 작용시켜 정미성이 있는 5'-뉴클레오티드류로 변환하는 공정을 갖는 발효 조미료의 제조 방법에 의해 해결한다.An object of the present invention is to provide a nucleic acid-containing soy sauce-style seasoning that contains a high concentration of nucleic acids only from fermentation-derived 5'-nucleotides without externally adding 5'-nucleotides, and a method for producing the same.
A step of heating and decomposing moromi with a salt concentration of 4% (w/v) or less by adding water or saline to a grain raw material containing soybean or wheat as a main raw material, preparing a solid yeast by inoculating koji bacteria, and preparing the moromi; A step of preparing a sterilized moromi by inactivating the phosphatase by heat treatment; a step of inoculating the sterilized moromi with a nucleic acid-producing microorganism to perform nucleic acid fermentation in a container capable of suppressing the incorporation of harmful microorganisms; A fermentation seasoning comprising a step of preparing an RNA extract by self-digesting the RNA in the cells to liberate the RNA from the moromi, and a step of converting the RNA extract into refined 5'-nucleotides by acting a nuclease and deaminase is solved by the manufacturing method of
Description
본 발명은 제조 공정 중에서 핵산 생산 미생물에 의해 생산된 핵산을 고농도로 함유하는 발효 조미료 및 그 제조 방법에 관한 것이다.The present invention relates to a fermented seasoning containing a nucleic acid produced by a nucleic acid producing microorganism in a high concentration during the manufacturing process and a method for producing the same.
간장은 「간장 품질 표시 기준」에 의해 「특급」 「상급」 「표준」으로 구별되어 있다. 이들 등급은 「감칠맛」의 지표라고 일컬어지고 있는 「질소분」의 함량이나 색도(색의 농담) 등으로 정해지고 있고, 일반적으로는, 질소분의 함유량이 많을수록 감칠맛이 풍부한 간장이라고 여겨지고 있다. 간장의 감칠맛에 기여하는 질소분으로서는, 글루타민산을 비롯한 아미노산류가 알려져 있다. Soy sauce is classified into "Exclusive", "Superior" and "Standard" according to the "Soy Sauce Quality Labeling Standard". These grades are determined by the content and chromaticity (shades of color), etc. of "nitrogen content", which are said to be indicators of "umami taste", and in general, the higher the nitrogen content, the richer the umami. Amino acids including glutamic acid are known as nitrogen content contributing to the umami taste of soy sauce.
한편, 아미노산 이외의 감칠맛 성분으로서, 5'-뉴클레오티드류가 알려져 있다. 5'-뉴클레오티드류란, 5'-아데닐산, 5'-구아닐산, 5'-이노신산 및 5'-크산틸산 등을 비롯한 정미성(呈味性) 뉴클레오티드류의 총칭이며, 이들을 포함하는 조미료는 핵산계 조미료라고 불리는 경우도 있다. 5'-뉴클레오티드류는 단독으로도 강한 정미 작용을 갖지만, 간장에 포함되는 L-글루타민산이 존재하면, 상승 효과에 의해 풍미를 현저히 개선 강화할 수 있는 것이 알려져 있다.On the other hand, 5'-nucleotides are known as umami components other than amino acids. 5'-nucleotides are a generic term for refined nucleotides including 5'-adenylic acid, 5'-guanylic acid, 5'-inosine acid and 5'-xanthyl acid, and seasonings including these are nucleic acids It is sometimes called system seasoning. Although 5'-nucleotides have a strong rice-polishing effect even alone, it is known that, when L-glutamic acid contained in soy sauce is present, the flavor can be remarkably improved and strengthened by a synergistic effect.
그러나, 간장 중에는 각종 미생물에서 유래하는 다량의 포스파타아제가 존재하고 있기 때문에, 간장에 정미성 뉴클레오티드류를 첨가해도, 뉴클레오티드류는 포스파타아제의 작용에 의해 탈인산화되어 정미성이 없는 뉴클레오시드로 분해된다.However, since a large amount of phosphatases derived from various microorganisms exist in the liver, even when refined nucleotides are added to the liver, the nucleotides are dephosphorylated by the action of the phosphatase, so that the nucleosides have no taste. is decomposed into
예컨대, 열처리 전의 생간장은 이 포스파타아제 활성이 특히 강한 것 중 하나이며, 첨가한 정미성 뉴클레오티드류는 첨가 후 하루에 거의 분해 소실되어, 이들 뉴클레오티드류의 정미성은 완전히 소실되어 버리는 것이 알려져 있다(특허문헌 1). 또한, 포스파타아제 활성은, 생간장에 상당히 강력히 존재하지만, 열처리(80℃ 도달 온도)한 간장에도, 생간장의 10~25% 정도 잔존하고 있는 것이 알려져 있다.For example, it is known that raw soy sauce before heat treatment is one of particularly strong phosphatase activity, and the added clean nucleotides are almost decomposed and lost within one day after addition, and the cleanliness of these nucleotides is completely lost ( Patent Document 1). In addition, although phosphatase activity exists quite strongly in raw soy sauce, it is known that about 10 to 25% of raw soy sauce remains even in soy sauce subjected to heat treatment (temperature reached at 80°C).
따라서, 포스파타아제 활성을 실활(失活)시키는 것이, 간장에 5'-뉴클레오티드류를 첨가하여, 안정적으로 유지하기 위해서는 필요하다고 되어 있다. 종래 알려져 있는 5'-뉴클레오티드류 함유 간장의 제조법은, 통상법에 의해 제조된 간장의 포스파타아제 활성을 실활시킨 후에 5'-뉴클레오티드류를 별도로 첨가하는 방법이었다(특허문헌 2, 3). 그러나, 간장에 핵산을 첨가한 경우에는, 식품 표시 관련 법규의 규정에 기초하여, 원재료에 첨가물의 표시가 필수가 되기 때문에, 「조미료(핵산)」라고 표시하지 않으면 안 된다. 이러한 간장풍 조미료는 「화학 조미료 무첨가」나, 「천연 양조」를 주장할 수 없어, 천연 지향, 자연 지향의 소비자에 대해서는 소구력이 약하였다.Therefore, it is said that inactivating the phosphatase activity is necessary in order to add 5'-nucleotides to the liver and maintain it stably. The conventionally known method for producing a 5'-nucleotide-containing soy sauce is a method of separately adding 5'-nucleotides after inactivating the phosphatase activity of the liver produced by a conventional method (Patent Documents 2 and 3). However, when nucleic acid is added to soy sauce, it must be labeled as “seasoning (nucleic acid)” because the labeling of the additive is essential to the raw material based on the provisions of laws and regulations related to food labeling. These soy sauce-style seasonings cannot claim "chemical seasoning-free" or "natural brewing", so they have weak appeal to natural-oriented and nature-oriented consumers.
정미성 뉴클레오티드류를 발효 생산하는 공지된 미생물로서, 예컨대, 고핵산형 효모 엑기스의 제조에 사용되고 있는 칸디다 유틸리스(Candida utilis)나 핵산 조미료의 제조에 이용되고 있는 코리네박테리움 글루타미컴(Corynebacterium glutamicum) 등이 알려져 있다. 만일 포스파타아제 활성이 없는 간장 혹은 모로미 중에서 이들 미생물을 생육시켜, 핵산을 발효 생산하고자 해도, 내염성이 없기 때문에, 간장이나 간장 모로미 중에서는 증식할 수 없어, 핵산을 고함유시킬 수 없다. 이러한 배경에서, 종래의 간장 제조 업계에서는, 5'-뉴클레오티드류를 첨가하지 않고, 간장 모로미나 압착 후의 생간장, 혹은 열처리 후의 간장 중에서 5'-뉴클레오티드류를 발효에 의해 생성 축적하는 것은 매우 곤란하다고 생각되고 있어, 지금까지 실용화되어 있지 않다.As a known microorganism for fermenting and producing refined nucleotides, for example, Candida utilis used for production of high nucleic acid type yeast extract or Corynebacterium glutamicum used for production of nucleic acid seasoning glutamicum ) are known. If these microorganisms are grown in liver or moromi without phosphatase activity and nucleic acid is fermented and produced, they cannot be grown in soy sauce or soy moromi because of the lack of salt resistance, and thus the nucleic acid content cannot be high. Against this background, in the conventional soy sauce manufacturing industry, without adding 5'-nucleotides, it is very difficult to generate and accumulate 5'-nucleotides by fermentation in raw soy sauce after compression or heat treatment. It is considered, and has not been put into practical use until now.
한편, 간장의 양조 공정 중에서의 직접적인 발효 생산은 아니지만, 정미 성분으로서 핵산을 함유하는 간장의 제조 방법은 몇 개의 문헌에 기재되어 있다. 예컨대, 특허문헌 4에는, 천연 양조 간장의 모로미의 제조 공정에 있어서, 출국(出麴)한 원료에 첨가하는 주입수로서, 청주 등의 발효 지게미에 가수(加水)하여 50~60℃에서 1~2주간 자기 소화시킨 것, 또는 이것을 압착한 정미성이 높은 액을 이용하는 것을 특징으로 하는 농후 간장의 제조법이 개시되어 있다. 그러나, 특수한 원료(즉 술지게미나 소주 증류 폐액)를 이용할 필요가 있다. 따라서, 통상의 양조 간장과 비교하면, 풍미도 상당히 떨어진 것이 되는 결점을 갖는다.On the other hand, although it is not a direct fermentation production in the brewing process of soy sauce, a manufacturing method of soy sauce containing a nucleic acid as a rice-polishing ingredient is described in several documents. For example, in patent document 4, in the manufacturing process of moromi of natural brewed soy sauce, as infusion water added to the raw material leaving the country, it is hydrolyzed to fermented lees, such as sake, and 1~ at 50-60 degreeC. A method for producing a thick soy sauce characterized by using a highly polished liquid obtained by self-digesting for 2 weeks or compressed therein is disclosed. However, it is necessary to use special raw materials (ie, sake lees or shochu distillation waste liquid). Therefore, compared with a normal brewed soy sauce, it has the fault that the flavor is also considerably inferior.
본 발명은 5'-뉴클레오티드류를 외부에서 첨가하지 않고, 발효 유래의 5'-뉴클레오티드류를 고농도로 함유하는 발효 조미료 및 그 제조 방법을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a fermentation seasoning containing 5'-nucleotides derived from fermentation at a high concentration without externally adding 5'-nucleotides, and a method for producing the same.
본 발명자들은, 상기 과제를 해결하기 위해서 예의 검토를 행한 결과, 대두 또는 밀 등을 주원료로 하는 곡물 원료에 국균(麴菌)을 접종하여 조제한 고체 누룩에, 무염 또는 저염의 주입수를 첨가하여 모로미를 조제하고, 이 모로미를 가열 살균(포스파타아제 실활) 후, 위해 미생물의 혼입을 억제할 수 있는 용기에서 오염을 방지하면서 핵산 발효를 행함으로써, 내염성을 갖지 않는 핵산 발효 생산 미생물에 의한 핵산 발효가 가능해져, 핵산을 별도로 첨가하지 않고, 발효 유래의 핵산을 고농도로 함유하는 발효 조미료가 얻어지는 것을 발견하고, 본 발명을 완성시켰다.The present inventors, as a result of earnestly examining in order to solve the above problem, added salt-free or low-salt infusion water to solid koji prepared by inoculating koji bacteria into grain raw materials containing soybean or wheat as a main raw material, and adding moromi , and then heat sterilize the moromi (phosphatase inactivation), and then perform nucleic acid fermentation while preventing contamination in a container capable of suppressing the incorporation of harmful microorganisms. became possible, and found that a fermented seasoning containing a nucleic acid derived from fermentation at a high concentration can be obtained without separately adding a nucleic acid, and completed the present invention.
즉, 본 발명은 핵산(단 첨가 유래의 핵산을 제외한다) 10 ppm(w/v) 이상과, HDMF 2 ppm(w/v) 이상을 함유하는 발효 조미료를 제공하는 것이다.That is, the present invention provides a fermented seasoning containing 10 ppm (w/v) or more of nucleic acids (excluding nucleic acids derived from addition) and 2 ppm (w/v) of HDMF.
또한, 본 발명은 대두 또는 밀 등을 주원료로 한 곡물 원료에, 국균을 접종하여 고체 누룩을 조제하고, 물 또는 식염수를 첨가하여 식염 농도 4%(w/v) 이하의 모로미를 가온 분해하는 공정과, 상기 모로미를 가열 처리하여 포스파타아제를 실활시킴으로써 살균 모로미를 조제하는 공정과, 상기 살균 모로미에 핵산 생산 미생물을 접종하여 위해 미생물의 혼입을 억제할 수 있는 용기에서 핵산 발효를 행하는 공정과, 상기 핵산 생산 미생물을 자기 소화하여 균체 내의 RNA를 모로미 중에 유리(遊離)시킴으로써 RNA 추출물을 조제하는 공정과, 상기 RNA 추출물에 리보뉴클레아제 및 아데닐산데아미나아제를 작용시켜 정미성이 있는 5'-뉴클레오티드류로 변환하는 공정을 갖는 발효 조미료의 제조 방법을 제공하는 것이다.In addition, the present invention is a process of heating and decomposing moromi with a salt concentration of 4% (w/v) or less by inoculating a grain raw material containing soybean or wheat as a main raw material, preparing a solid yeast, and adding water or saline A step of preparing a sterilized moromi by heat-treating the moromi to inactivate the phosphatase, and inoculating the sterilized moromi with a nucleic acid-producing microorganism to perform nucleic acid fermentation in a container capable of suppressing incorporation of harmful microorganisms; A step of preparing an RNA extract by self-digesting the nucleic acid-producing microorganism to liberate the RNA in the microbial cells from the moromi, and 5' with ribonuclease and adenylate deaminase to act on the RNA extract - To provide a method for producing a fermented seasoning having a step of converting it into nucleotides.
본 발명에 의하면, 5'-뉴클레오티드류(핵산)를 외부에서 첨가하지 않고, 핵산 생산 미생물이 발효 중에 생산하는 5'-뉴클레오티드류만으로 핵산을 고농도로 함유하는 발효 조미료 및 그 제조 방법을 제공할 수 있다.According to the present invention, without adding 5'-nucleotides (nucleic acids) from the outside, only 5'-nucleotides produced by a nucleic acid-producing microorganism during fermentation can provide a fermentation seasoning containing nucleic acids in a high concentration and a method for producing the same. there is.
도 1은 본 실시형태에 따른 핵산 함유 발효 조미료의 제조 방법의 공정도이다.
도 2는 무염 모로미를 가열한 후에 정미성 핵산(5'-구아닐산 및 5'-이노신산)을 첨가하고, 경시적으로 핵산 농도를 정량한 결과를 도시한 도면이다.1 is a flowchart of a method for producing a nucleic acid-containing fermented seasoning according to the present embodiment.
FIG. 2 is a view showing the results of quantifying the nucleic acid concentration over time after heating unsalted moromi after adding refined nucleic acids (5'-guanylic acid and 5'-inosinic acid).
이하, 본 발명에 따른 발효 조미료 및 그 제조 방법을 상세히 설명한다. 도 1은 본 실시형태에 따른 핵산 함유 발효 조미료의 제조 방법의 공정도이다.Hereinafter, a fermented seasoning according to the present invention and a manufacturing method thereof will be described in detail. 1 is a flowchart of a method for producing a nucleic acid-containing fermented seasoning according to the present embodiment.
본 발명에 있어서, 핵산이란, 5'-아데닐산, 5'-구아닐산, 5'-이노신산 및 5'-크산틸산 등을 비롯한 정미성 뉴클레오티드류를 의미한다.In the present invention, the term nucleic acid refers to refined nucleotides including 5'-adenylic acid, 5'-guanylic acid, 5'-inosine acid, 5'-xanthyl acid, and the like.
본 발명의 실시형태에 있어서는, 먼저 곡물 원료로부터 조제한 고체 누룩에 물 또는 식염수를 혼화하여, 식염 농도 4%(w/v) 이하, 바람직하게는 식염 농도 4%(w/v) 미만, 보다 바람직하게는 식염 농도 0%(w/v)의 간장 모로미를 조제하고, 25~57℃에서 0~48시간 가온 분해한다. 바람직하게는, 일본 특허 제3827300호 기재와 같이, 70~80℃의 열수 또는 식염수를 고체 누룩과 혼화하고, 50~57℃로 모로미 온도를 유지한 채로, 탱크 내에서 간헐적 또는 연속적으로 교반하며, 15~30시간 효소 분해하는 것이 바람직하다.In the embodiment of the present invention, first, water or saline is mixed with solid koji prepared from grain raw materials, and the salt concentration is 4% (w/v) or less, preferably the salt concentration is less than 4% (w/v), more preferably Soy sauce moromi with a salt concentration of 0% (w/v) is prepared and decomposed by heating at 25-57°C for 0-48 hours. Preferably, as described in Japanese Patent No. 3827300, hot water or saline at 70 to 80 ° C. is mixed with solid yeast, and while maintaining the Moromi temperature at 50 to 57 ° C., intermittently or continuously stirring in a tank, Enzymatic digestion for 15 to 30 hours is preferable.
여기서, 곡물 원료란, 예컨대 환대두(丸大豆), 탈지 대두, 대두 단백, 밀 글루텐, 완두콩, 잠두콩, 팥 등으로 대표되는 단백질 원료와, 밀, 보리, 호밀, 밀기울, 쌀, 쌀겨，옥수수, 전분박(澱粉粕) 등으로 대표되는 전분질 원료를 가리킨다. 이들은 단독으로, 또는 조합하여 이용할 수 있다.Here, the grain raw material includes, for example, a protein raw material represented by round soybean, defatted soybean, soy protein, wheat gluten, pea, broad bean, red bean, and the like, wheat, barley, rye, wheat bran, rice, rice bran, corn , refers to starchy raw materials represented by starch foil and the like. These can be used individually or in combination.
여기서 이용되는 누룩(고체 누룩)은, 통상법에 의해 원료 처리된 단백질 원료 또는 이것에 전분질 원료를 혼합한 것에, 아스페르길루스 소예(Aspergillus sojae), 아스페르길루스 오리제(Aspergillus oryzae)로 대표되는 국균을 접종하고, 2~3일 고체 배양(제국(製麴))함으로써 얻어진다. 단백질 원료에 전분질 원료를 혼합하는 경우, 배합 비율은 특별히 한정하는 것은 아니지만, 예컨대 통상의 간장에 가까운 조미료를 얻고자 하는 경우, 중량비로 1:0.25~4로 하는 것이 바람직하다.The koji (solid koji) used here is a protein raw material processed by a conventional method or a starch raw material mixed with it, aspergillus sojae , Aspergillus oryzae , represented by It is obtained by inoculating the Kukbacterium to be used and performing solid culture (imperialization) for 2-3 days. When the starch raw material is mixed with the protein raw material, the blending ratio is not particularly limited. For example, when obtaining a seasoning close to normal soy sauce, it is preferably 1:0.25 to 4 by weight.
주입에 이용하는 물 또는 식염수는, 누룩이 충분히 잠기는 정도이면 되고, 일반적으로는, 누룩 중량에 대해 1~10 용량배(v/w)로 하는 것이 바람직하다. 또한, 가온 분해 시에, 방미성이나 분해 효율의 향상, 풍미 향상을 위해서 후술하는 식용의 산이나 효소제, 활성탄을 첨가해도 좋다.The water or saline used for injection should just be enough to fully immerse the koji, and in general, it is preferable to set it as 1-10 volume times (v/w) with respect to the koji weight. In addition, at the time of decomposition by heating, you may add an edible acid, an enzyme agent, and activated carbon, which will be described later, in order to improve anti-mildness, decomposition efficiency, and flavor.
가온 분해 시에는, 모로미의 분해 촉진을 위해서, 효소제를 최종 농도가 0.001~1%(w/v)가 되도록 첨가해도 좋다. 효소제로서는, 예컨대, 프로테아제(엔도프로테아제, 엑소프로테아제), 셀룰라아제, 펙티나아제 등을 들 수 있다.In the case of thermal decomposition, in order to accelerate the decomposition of moromi, an enzyme agent may be added so that the final concentration is 0.001 to 1% (w/v). As an enzyme agent, a protease (endoprotease, exoprotease), a cellulase, a pectinase, etc. are mentioned, for example.
다음으로 가온 분해를 행한 식염 농도 4%(w/v) 이하의 고액(固液) 분리 전의 간장 모로미에 대해 가열 처리를 행하여, 살균 모로미를 조제한다. 이 가열 처리에 의해, 전술한 제국 공정에 있어서 국균이 생산한 포스파타아제 등의 핵산 분해 효소를 실활시키고, 모로미의 살균을 행한다. 여기서, 살균기의 부하 경감이나, 핵산 생산 미생물의 생육 속도 향상을 목적으로 하여, 살균 전에 적절히 가수를 행하여 모로미를 희석해도 좋다.Next, the soy sauce moromi before the solid-liquid separation with a salt concentration of 4% (w/v) or less subjected to thermal decomposition is heat-treated to prepare a sterilized moromi. By this heat treatment, nucleolytic enzymes, such as phosphatase, which were produced by the domestic bacteria in the above-mentioned imperial step are inactivated, and the moromi is sterilized. Here, for the purpose of reducing the load on the sterilizer or improving the growth rate of the nucleic acid-producing microorganism, water may be appropriately diluted before sterilization to dilute the moromi.
여기서 가열 처리의 방법은 특별히 한정되지 않고, UHT, HTST, 레토르트, 가압 탱크, 스팀 인젝션, 스팀 인퓨전, 오토클레이브, 플레이트 히터, 표면 긁기식, 줄식 열교환, 튜블러식 열교환 등의 가열 처리 방법의 어느 것을 이용해도 좋으나, 바람직하게는 가압 탱크나 튜블러식 열교환기를 이용하는 것이 좋다. 예컨대, 모로미를 가압 탱크에 넣고, 균일하게 교반하면서 가압 가온하는 것 등으로 핵산 분해 효소의 실활이나 살균을 행할 수 있다. 가열 온도가 지나치게 낮거나, 혹은 가열 시간이 지나치게 짧으면, 핵산 분해 효소의 실활이나 잡균의 살균이 불충분해지기 때문에 바람직하지 않다. 반대로, 가열 온도가 지나치게 높거나, 혹은 가열 시간이 지나치게 길면, 조미료의 풍미가 열화되기 때문에 바람직하지 않다. 선택하는 가열 방법에 따라 최적 조건은 상이하지만, 예컨대, 80℃에서는 2분 이상 180분 이하, 121℃에서는 5초 이상 15분 이하, 130℃에서는 1초 이상 30초 이하가 바람직하다.The method of heat treatment is not particularly limited here, and any of heat treatment methods such as UHT, HTST, retort, pressurized tank, steam injection, steam infusion, autoclave, plate heater, surface scraping type, file type heat exchange, and tubular type heat exchange. It may be used, but it is preferable to use a pressurized tank or a tubular heat exchanger. For example, nucleolytic enzyme inactivation or sterilization can be performed by placing Moromi in a pressurized tank and heating under pressure while uniformly stirring. When the heating temperature is too low or the heating time is too short, it is not preferable because the deactivation of the nucleolytic enzyme and the sterilization of various bacteria become insufficient. Conversely, when the heating temperature is too high or the heating time is too long, the flavor of the seasoning is deteriorated, which is not preferable. Although optimal conditions differ depending on the heating method selected, for example, 2 minutes or more and 180 minutes or less at 80 degreeC, 5 seconds or more and 15 minutes or less at 121 degreeC, and 1 second or more and 30 seconds or less at 130 degreeC are preferable.
조제한 모로미는, 방미성의 향상이나 맛의 조정을 위해서, pH 조정을 행해도 좋다. 조정 후의 pH는, 방미성과 효모의 발효성의 관점에서, 3.0~7.0, 바람직하게는 4.0~5.5로 하는 것이 바람직하다. pH 조정의 타이밍으로서는, 가온 분해 시, 모로미의 가열 처리 전, 모로미의 가열 처리 후의 어느 쪽이어도 좋다. pH 조정제로서의 식용의 산으로서는, 예컨대, 젖산, 아세트산, 말산, 시트르산, 글루콘산, 아디프산 등을 들 수 있으나, 풍미의 점에서, 젖산이 바람직하다.The prepared moromi may perform pH adjustment for the improvement of anti-fouling property and adjustment of taste. The pH after adjustment is 3.0-7.0 from a viewpoint of anti-mold property and the fermentability of yeast, It is preferable to set it as 4.0-5.5 preferably. The timing of pH adjustment may be either before heat treatment of moromi or after heat treatment of moromi during thermal decomposition. Examples of the edible acid as the pH adjuster include lactic acid, acetic acid, malic acid, citric acid, gluconic acid, and adipic acid. From the viewpoint of flavor, lactic acid is preferable.
모로미에는 핵산 생산 미생물에 의한 핵산 발효를 원활히 행하기 위해서, 당질을 0~20%(w/v) 첨가해도 좋다. 예컨대, 글루코오스, 프룩토오스, 수크로오스, 말토오스, 만노오스, 글리세롤 등, 핵산 생산 미생물이 자화(資化)할 수 있는 당질을 이용할 수 있으나, 자화 효율을 생각하면 글루코오스의 사용이 바람직하다. 또한, 이들 당을 포함하는 식품 소재, 예컨대, 설탕, 포도당 과당 액당, 과당 포도당 액당, 삼온당(三溫糖), 당밀 등을 이용해도 좋다.In order to smoothly perform nucleic acid fermentation by nucleic acid producing microorganisms, 0 to 20% (w/v) of carbohydrates may be added to Moromi. For example, glucose, fructose, sucrose, maltose, mannose, glycerol, etc., can be used as a carbohydrate that can be magnetized by a nucleic acid-producing microorganism, but glucose is preferably used in consideration of magnetization efficiency. Moreover, you may use the food material containing these sugars, for example, sugar, glucose-fructose-fructose liquid sugar, fructose-glucose-glucose liquid sugar, triple sugar, molasses, etc.
또한, 핵산 발효 전에도, 모로미의 분해 촉진이나 압착성의 향상을 위해서, 효소제를 최종 농도가 0.001~1%(w/v)가 되도록 첨가해도 좋다. 효소제로서는, 예컨대, 프로테아제(엔도프로테아제, 엑소프로테아제), 셀룰라아제, 펙티나아제 등을 들 수 있다.In addition, even before nucleic acid fermentation, an enzyme agent may be added so that the final concentration is 0.001 to 1% (w/v) in order to promote decomposition of moromi and improve compressibility. As an enzyme agent, a protease (endoprotease, exoprotease), a cellulase, a pectinase, etc. are mentioned, for example.
또한, 핵산 발효 전에, 쓴맛을 제거하여 풍미를 향상시키기 위해서, 모로미에 활성탄을 첨가해도 좋다. 활성탄은 분말인 것이 바람직하고, 평균 입자 직경이 10~100 ㎛인 것을 사용하는 것이 보다 바람직하다. 활성탄의 첨가량은, 모로미 원료에 대해 0.1~5%(w/w)인 것이 바람직하다. 활성탄의 종류는 용도에 따라 적절히 선택할 수 있고, 예컨대, 쓴맛 제거, 악취 제거, 맛의 조정, 색의 조정 또는 이들 기능을 갖는 활성탄을 조합하여 사용할 수 있다.In addition, before nucleic acid fermentation, activated carbon may be added to moromi in order to remove bitterness and improve flavor. It is preferable that the activated carbon is a powder, and it is more preferable to use the thing with an average particle diameter of 10-100 micrometers. It is preferable that the addition amount of activated carbon is 0.1 to 5% (w/w) with respect to the moromi raw material. The kind of activated carbon can be appropriately selected according to the use, and for example, bitter taste removal, odor removal, taste adjustment, color adjustment, or activated carbon having these functions can be used in combination.
또한, 핵산 발효 전에, 상기 살균 모로미를 고액 분리해도 좋다. 고액 분리의 방법은 특별히 한정되지 않고, 예컨대, 압착, 원심 분리, 여과 등의 방법으로 실시할 수 있다.In addition, before the nucleic acid fermentation, the sterilized moromi may be separated into solid and liquid. The method of solid-liquid separation is not specifically limited, For example, it can implement by methods, such as crimping|compression-bonding, centrifugation, and filtration.
본 발명에서 사용하는 핵산 생산 미생물은, 핵산을 생산할 수 있는 미생물이면 특별히 제한되지 않고, 예컨대 칸디다 유틸리스(Candida utilis), 사카로마이세스 세레비시에(Saccharomyces cerevisiae), 자이고사카로마이세스 룩시이(Zygosaccharomyces rouxii), 스키조사카로마이세스 폼베(Schizosaccharomyces pombe), 클루이베로마이세스 마르시아누스(Kluyveromyces marxianus) 등의 효모, 코리네박테리움 글루타미컴(Corynebacterium glutamicum), 코리네박테리움 암모니아게네스(Corynebacterium ammoniagenes), 바실러스 서브틸리스(Bacillus subtilis) 등의 세균, 또는 이들의 영양 요구·아날로그 내성 변이주가 이용된다. 본 발명의 모로미는 무염 또는 저염이기 때문에, 본래, 내염성이 약한 효모나 세균을 사용할 수 있다.The nucleic acid-producing microorganism used in the present invention is not particularly limited as long as it is a microorganism capable of producing a nucleic acid, for example, Candida utilis ( Candida utilis ), Saccharomyces cerevisiae cerevisiae ), Zygosaccharomyces rouxii ), Schizosaccharomyces pombe ), Kluyveromyces marcianus ( Kluyveromyces ) marxianus ), such as yeast, Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium ammoniagenes , Bacillus subtilis , such as bacteria, or their nutritional requirements/analog resistance mutants are used. Since the moromi of the present invention is salt-free or low-salt, yeast or bacteria that are inherently weak in salt resistance can be used.
이들 핵산 생산 미생물의 첨가 농도는, 효모이면, 모로미 1 g당 1×104개 이상, 바람직하게는 1×105~1×107개가 되도록 첨가하는 것이 바람직하고, 세균이면, 모로미 1 g당 1×104개 이상, 바람직하게는 1×105~1×107개가 되도록 첨가하는 것이 바람직하다.The concentration of these nucleic acid-producing microorganisms added is preferably 1×10 4 or more, preferably 1×10 5 to 1×10 7 , per 1 g of moromi in the case of yeast, and in the case of bacteria, per 1 g of moromi It is preferable to add 1×10 4 or more, preferably 1×10 5 to 1×10 7 .
모로미는 위해 미생물의 혼입을 억제할 수 있는 용기에 투입되고, 이 용기 내에서 핵산 발효를 행한다. 여기서, 위해 미생물의 혼입을 억제할 수 있는 용기란, 용기 내부와 외기를 차단할 수 있는 구조를 갖는 것이면 되고, 실험적으로는 폴리프로필렌제의 멸균이 완료된 입구가 큰 병이나 유리제의 미디어 보틀 등을 사용할 수 있고, 공업적으로는 용기 내에 제균된 공기를 공급할 수 있는 기능을 갖는 자 퍼멘터나 가압식의 발효 탱크 등을 사용할 수 있다. 또한, 공기의 제균에는, 0.3 ㎛ 이상의 먼지를 99.97% 이상 집진할 수 있는 필터, 예컨대 HEPA 필터 등을 이용할 수 있다.Moromi is put into a container capable of suppressing the incorporation of harmful microorganisms, and nucleic acid fermentation is performed in this container. Here, a container capable of inhibiting the incorporation of harmful microorganisms should just have a structure that can block the inside and outside air of the container. Industrially, it is possible to use a self-fermenter or a pressurized fermentation tank having a function of supplying sterilized air into the container. In addition, a filter capable of collecting 99.97% or more of dust of 0.3 µm or larger, such as a HEPA filter, can be used for air sterilization.
핵산 발효는, 핵산 생산 미생물을 생육할 수 있는 온도, 구체적으로는 15~45℃, 바람직하게는 20~35℃에서 1~90일, 바람직하게는 2~28일 행한다.Nucleic acid fermentation is performed at a temperature at which a nucleic acid-producing microorganism can grow, specifically 15 to 45°C, preferably 20 to 35°C, for 1-90 days, preferably 2-28 days.
본 실시형태에 따른 발효 조미료에 포함되는 알코올은, 핵산 발효 공정에서 첨가하는 당 농도나 사용하는 미생물의 종류나 온도 조건에 의해 0~20%(w/v)로 생산량을 조정할 수 있고, 또한, 핵산 발효 종료 시에 알코올을 첨가해도 좋으나, 발효 조미료로서 양호한 풍미를 갖게 하기 위해서, 바람직하게는 8%(w/v) 미만, 보다 바람직하게는 2~7%(w/v)로 하는 것이 좋다.The alcohol contained in the fermentation seasoning according to the present embodiment can adjust the production amount to 0 to 20% (w/v) depending on the sugar concentration added in the nucleic acid fermentation process, the type of microorganism used, and temperature conditions, Alcohol may be added at the end of nucleic acid fermentation, but in order to have a good flavor as a fermented seasoning, it is preferably less than 8% (w/v), more preferably 2 to 7% (w/v). .
핵산 발효의 종료 후에는, 모로미 중의 핵산 생산 미생물을 자기 소화시키거나, 혹은 프로테아제나 β-글루카나아제를 포함하는 효소제를 이용하여 효모 세포벽을 용해시켜, 균체 내의 RNA를 모로미 중에 유리시킨다. 자기 소화는 모로미를 일정 시간 가온함으로써 행할 수 있다. 자기 소화는 예컨대 핵산 생산 미생물의 RNA의 용출이 최대가 되는 데 충분한 조건, 예컨대 품온(品溫) 40~60℃에서 1분간~24시간 행하는 것이 보다 바람직하다. 품온이 40℃ 미만에서는 자기 소화에 시간을 필요로 하고, 또한 모로미 액즙에 대한 핵산 생산 미생물 유래의 RNA의 용출이 충분하지 않으며, 반대로 60℃를 초과하는 온도에서는 모로미의 가열 열화에 의한 갈변이나 풍미의 악화를 초래하기 때문에 바람직하지 않다. 시간이 1분간 미만에서는 핵산 생산 미생물 유래의 RNA의 용출이 충분하지 않고, 반대로 24시간을 초과하면 가열 열화에 의해 모로미의 갈변이나 풍미의 악화를 초래하기 때문에 바람직하지 않다. 또한 자기 소화 시에 핵산의 유리 효율을 향상시킬 목적으로, 소화 전에 내재성의 핵산 분해 효소를 가열 실활시키는 방법이나, 동결 융해를 행하는 방법, 에탄올 등의 유기 용매를 첨가하는 방법, 초음파 처리를 행하는 방법, pH 4~6 정도의 산성 혹은 pH 8~12 정도의 알칼리성으로 조정하는 방법 등, 공지된 방법을 조합하여 사용할 수도 있다. 또한, 효모 세포벽을 용해하는 효소제로서는, 투니카제(상표, 아마노 엔자임사 제조), 데나짐 GEL(상표, 나가세 켐텍스사 제조), 필트라제 BRX(상표, DSM사 제조) 등을 정법에 따라 사용할 수 있다.After completion of the nucleic acid fermentation, the nucleic acid-producing microorganisms in the moromi are self-digested, or the yeast cell wall is lysed using an enzyme agent containing protease or β-glucanase to liberate the RNA in the microbial cells in the moromi. Self-extinguishing can be performed by heating the moromi for a certain period of time. It is more preferable that the self-digestion be performed, for example, under conditions sufficient to maximize the elution of RNA of the nucleic acid-producing microorganism, for example, at a temperature of 40 to 60° C. for 1 minute to 24 hours. When the product temperature is less than 40°C, self-digestion takes time, and the elution of RNA derived from the nucleic acid-producing microorganism into the moromi juice is not sufficient. It is undesirable because it causes deterioration of When the time is less than 1 minute, the elution of RNA derived from the nucleic acid-producing microorganism is not sufficient, and conversely, when it exceeds 24 hours, browning of the moromi and deterioration of flavor are caused by heat deterioration, which is not preferable. In addition, for the purpose of improving the efficiency of nucleic acid liberation during self-digestion, a method of inactivating an endogenous nucleolytic enzyme by heating before digestion, a method of freeze-thawing, a method of adding an organic solvent such as ethanol, a method of performing sonication , a method of adjusting to acidity of about pH 4-6 or alkalinity of about pH 8-12 can also be used combining well-known methods. In addition, as an enzyme agent for dissolving the yeast cell wall, Tunikase (trademark, manufactured by Amano Enzyme), Denazim GEL (trademark, manufactured by Nagase Chemtex), Piltraze BRX (trademark, manufactured by DSM), etc. can be used
핵산 생산 미생물의 자기 소화 또는 효소 소화를 행한 후, 정미성이 없는 RNA 추출물에 효소 처리를 행하여 정미성이 있는 5'-뉴클레오티드류(핵산)로 변환한다. 효소는 뉴클레아제를 사용할 수 있고, 이에 더하여 데아미나아제를 사용하는 것이 바람직하다. 뉴클레아제로서는 리보뉴클레아제, 데옥시리보뉴클레아제 등을 들 수 있고, 리보뉴클레아제인 것이 바람직하다. 또한, 리보뉴클레아제 중에서도 절단 양식에 따라 P1 뉴클레아제, S1 뉴클레아제 등을 들 수 있고, 어느 쪽의 효소도 사용할 수 있다. 또한, 데아미나아제로서는 아데닐산데아미나아제인 것이 바람직하다. 이들 효소는 모두 시판의 각종 효소를 사용할 수 있다. 또는 맥아 혹은 발아미 등, 이들 효소를 포함하는 식품을 첨가할 수도 있다. 효소 처리에 있어서의 온도, pH, 첨가량, 처리 시간 등은, 사용하는 효소의 종류, 활성의 강도, 원하는 핵산 농도 등을 고려하여 적절히 결정할 수 있다. 또한, 효소 처리를 실시하는 순서는 뉴클레아제 처리물에 대해 데아미나아제 처리가 가능하면 되고, 뉴클레아제 처리를 실시한 후의 RNA 추출물에 데아미나아제를 첨가하여 데아미나아제 처리를 행해도 좋고, 이용하는 뉴클레아제와 데아미나아제가 함께 동일한 온도에서 효소 활성을 나타내는 경우에는, RNA 추출물에 뉴클레아제와 데아미나아제를 함께 첨가하여 동시에 효소 처리를 행해도 좋다.After self- or enzymatic digestion of a nucleic acid-producing microorganism, an unpolished RNA extract is subjected to enzymatic treatment to convert it into clean 5'-nucleotides (nucleic acids). The enzyme may use a nuclease, and in addition to this, it is preferable to use a deaminase. A ribonuclease, a deoxyribonuclease, etc. are mentioned as a nuclease, It is preferable that it is a ribonuclease. Moreover, among ribonucleases, P1 nuclease, S1 nuclease, etc. are mentioned depending on a cleavage mode, Any enzyme can be used. Moreover, it is preferable that it is adenylate deaminase as a deaminase. For all of these enzymes, various commercially available enzymes can be used. Alternatively, food containing these enzymes, such as malt or germinated rice, may be added. The temperature, pH, addition amount, treatment time, etc. in the enzyme treatment can be appropriately determined in consideration of the type of enzyme to be used, the strength of activity, the desired nucleic acid concentration, and the like. In addition, as for the procedure for performing the enzyme treatment, deaminase treatment may be performed on the nuclease-treated product, and deaminase treatment may be performed by adding deaminase to the RNA extract after nuclease treatment, When both the nuclease and the deaminase to be used exhibit enzymatic activity at the same temperature, the nuclease and the deaminase may be added to the RNA extract to perform the enzymatic treatment at the same time.
예컨대, 불용물이 제거된 RNA 추출물을 pH 4.0 이상, pH 7 미만, 바람직하게는 pH 4.5~6.0으로 조정한 후, 뉴클레아제를 첨가하여 60~80℃에서 30분간~12시간 뉴클레아제 처리한 후, 데아미나아제를 첨가하여 40~60℃에서 30분간~6시간 데아미나아제 처리를 행함으로써, 이노신산과 구아닐산을 충분히 생성할 수 있다.For example, after adjusting the RNA extract from which insoluble matter has been removed to pH 4.0 or more, pH less than 7, preferably pH 4.5 to 6.0, nuclease treatment at 60 to 80° C. for 30 minutes to 12 hours by adding nuclease After that, by adding deaminase and performing a deaminase treatment at 40 to 60° C. for 30 minutes to 6 hours, inosinic acid and guanylic acid can be sufficiently produced.
효소 처리 공정이 종료된 모로미는, 그대로 압착하지 않고 모로미(된장·페이스트)풍 조미료로 하면, 발효 유래의 핵산을 고농도로 포함하는 모로미풍 조미료로 할 수 있다. 또한, 모로미를 압착, 열처리, 청징, 여과 등의 통상법에 의한 처리를 행함으로써, 발효 유래의 핵산을 고농도로 포함하는 발효 조미료(예컨대, 간장풍 조미료)를 얻을 수 있다.If the moromi after the enzymatic treatment process is completed is used as a moromi (miso/paste)-style seasoning without compression as it is, a moromi-style seasoning containing a high concentration of nucleic acids derived from fermentation can be obtained. In addition, by subjecting the moromi to a conventional method such as compression, heat treatment, clarification, filtration, etc., it is possible to obtain a fermented seasoning (eg, soy sauce-style seasoning) containing a nucleic acid derived from fermentation at a high concentration.
효소 처리 공정이 종료된 후, 모로미에 식염을 첨가함으로써, 식염 함유 발효 조미료를 조제할 수도 있다. 즉, 모로미의 핵산 발효는 무염 또는 저염 조건하에서 행함으로써 핵산 발효를 촉진시키고, 이에 의해 얻어진 발효 모로미에 임의의 농도의 식염을 첨가함으로써, 간장다운 풍미를 갖는 발효 조미료를 제조할 수 있다. 식염의 첨가량은 적절히 설정할 수 있고, 식염 농도에 의해, 감염(減鹽) 간장풍 조미료나 진간장풍 조미료를 용이하게 조제할 수 있다.A salt-containing fermented seasoning can also be prepared by adding salt to the moromi after the enzyme treatment step is completed. That is, nucleic acid fermentation of Moromi is carried out under salt-free or low-salt conditions to promote nucleic acid fermentation, and by adding salt of an arbitrary concentration to the fermented Moromi obtained thereby, a fermented seasoning having a flavor similar to soy sauce can be produced. The amount of salt to be added can be set appropriately, and an infected soy sauce-style seasoning or a strong soy sauce-style seasoning can be easily prepared according to the salt concentration.
본 실시형태의 발효 조미료는, 첨가 유래의 핵산을 제외한 핵산 농도가 10 ppm 이상이다. 핵산 농도는 핵산 발효 공정에서 첨가하는 당 농도나 사용하는 미생물의 종류나 온도 조건에 의해 적절히 조정할 수 있으나, 핵산 농도는 핵산 생산 미생물의 핵산 생산 능력에 의존하기 때문에, 상한값은 자연히 핵산 생산 미생물의 종류에 따라 결정된다. 예컨대, 전술한 핵산 생산 효모로 핵산 발효를 행한 경우에는 5000 ppm 정도가 상한값이 된다. 한편, 핵산 발효 후에 가열 감압 농축, 스프레이 드라이, 동결 건조 등의 공지된 방법에 의해 농축 가공을 행하여, 발효 조미료 중의 핵산 농도를 상기 상한값 이상으로 높이는 것도 가능하다.The fermented seasoning of the present embodiment has a nucleic acid concentration of 10 ppm or more excluding the nucleic acid derived from the addition. The nucleic acid concentration can be appropriately adjusted depending on the sugar concentration added in the nucleic acid fermentation process, the type of microorganism used, or the temperature condition. is determined according to For example, when nucleic acid fermentation is performed with the above-mentioned nucleic acid producing yeast, about 5000 ppm becomes an upper limit. On the other hand, it is also possible to increase the concentration of the nucleic acid in the fermented seasoning to at least the upper limit value by performing concentration processing by a known method such as heating under reduced pressure concentration, spray drying, or freeze drying after nucleic acid fermentation.
일반적인 진간장의 핵산 함량이 검출 한계 이하인 데 대해, 본 실시형태의 발효 조미료는 상기한 바와 같이 현저한 양의 핵산을 함유하고 있다. 그 때문에, 간장에 원래 포함되는 글루타민산(0.2~2.0%(w/v) 정도)과의 상승 효과에 의해, 본 실시형태에 따른 발효 조미료 및 그것을 이용한 음식품의 감칠맛·깊은맛이 현저히 향상된다. 만일 핵산을 외부에서 첨가한 경우에는, 원재료에 첨가물의 표시가 필수가 되기 때문에, 화학 조미료 무첨가나, 천연 양조를 주장할 수 없으나, 본 실시형태의 발효 조미료이면 원재료명에 첨가물의 표시가 불필요하기 때문에, 자연 지향, 천연 지향의 소비자에 대해 부가 가치를 소구할 수 있다.While the nucleic acid content of general soy sauce is below the detection limit, the fermented seasoning of the present embodiment contains a significant amount of nucleic acid as described above. Therefore, by the synergistic effect with glutamic acid (about 0.2-2.0% (w/v)) originally contained in soy sauce, the umami and deep taste of the fermented seasoning which concerns on this embodiment and food-drinks using the same improves remarkably. If nucleic acid is added externally, since labeling of additives is essential to raw materials, it cannot be claimed that there is no chemical seasoning or natural brewing. Therefore, it is possible to appeal for added value to nature-oriented and natural-oriented consumers.
본 실시형태에 따른 발효 조미료는 또한, 향기 성분의 하나인 4-히드록시-2,5-디메틸-3(2H)-퓨라논(히드록시 디메틸 퓨라논, HDMF라고도 불린다) 농도가 2 ppm(w/v) 이상이고, 보다 바람직한 풍미를 갖고 있다. HDMF는 대표적인 퓨라논 화합물이고, 설탕풍의 단맛과 프루티함을 겸비하며, 간장 등, 정미 강화에 기여하는 조미료의 대표적 향기 성분으로서 알려져 있다. 본 발명의 발효 조미료는, 일반적인 진간장과 동등 이상의 HDMF를 함유하고 있고, 간장과 같은 향기로운 풍미가 우수한 것이 특징이다. HDMF 농도가 2 ppm보다 낮으면, 간장과 같은 향기로운 풍미가 불충분해져 버린다. 본 실시형태에 따른 발효 조미료에 포함되는 HDMF는, 원료인 누룩으로부터 발효를 거쳐 생성된 것이며, 전술한 핵산과 마찬가지로, 원재료명에 첨가물의 표시가 불필요하기 때문에, 자연 지향, 천연 지향의 소비자에 대해 부가 가치를 소구할 수 있다.The fermentation seasoning according to the present embodiment also has a concentration of 2 ppm (w /v) or more, and has a more preferable flavor. HDMF is a representative furanone compound, has a sugary sweetness and a fruity taste, and is known as a representative fragrance component of seasonings such as soy sauce that contributes to enhancing rice polishing. The fermented seasoning of the present invention contains HDMF equal to or higher than that of general soy sauce, and is characterized by excellent fragrant flavor similar to that of soy sauce. When the HDMF concentration is lower than 2 ppm, the fragrant flavor such as soy sauce becomes insufficient. HDMF contained in the fermented seasoning according to the present embodiment is produced through fermentation from koji, which is a raw material, and as with the above-mentioned nucleic acid, it is unnecessary to indicate an additive in the name of the raw material. You can claim added value.
본 실시형태의 다른 실시형태로서, 통상의 방법으로 조제한 생간장 또는 열처리 간장을 전기 투석 등으로 탈염하여 식염 농도를 낮춤으로써 식염 농도 4%(w/v) 이하의 탈염 생간장 또는 탈염 열처리 간장으로 하고, 그 후에는 전술한 바와 같이 가열 살균(포스파타아제 실활), 무균적인 핵산 발효, 정미성 핵산으로의 변환을 실시해도 좋다.As another embodiment of this embodiment, desalted raw soy sauce or heat-treated soy sauce prepared by a conventional method is desalted by electrodialysis or the like to lower the salt concentration to obtain desalted raw soy sauce or desalted heat-treated soy sauce with a salt concentration of 4% (w/v) or less. After that, as described above, heat sterilization (phosphatase inactivation), aseptic nucleic acid fermentation, and conversion to pure nucleic acid may be performed.
즉, 통상의 방법으로 조제한 생간장 또는 열처리 간장을 전기 투석 등으로 탈염하여 식염 농도를 낮춤으로써 식염 농도 4%(w/v) 이하의 탈염 생간장 또는 탈염 열처리 간장을 조제한다. 다음으로, 탈염 생간장 또는 탈염 열처리 간장을 재킷 부착 탱크에서 가열하거나, 플레이트 히터 또는 튜브 히터 등의 열교환기에서 가열 처리함으로써, 탈염 생간장 또는 탈염 열처리 간장 중의 포스파타아제를 실활시키고 탈염 생간장 또는 탈염 열처리 간장을 살균한다. 그리고, 살균한 탈염 생간장 또는 탈염 열처리 간장을 위해 미생물의 혼입을 억제할 수 있는 용기에 무균적으로 옮기고, 핵산 생산 미생물을 식균(植菌)하며, 앞서 설명한 발효 조건으로 핵산 발효를 행한다. 그 후 핵산 생산 미생물을 자기 소화하여 균체 내의 RNA를 모로미 중에 유리시킴으로써 RNA 추출물을 조제하고, RNA 추출물에 리보뉴클레아제 등의 뉴클레아제와 아데닐산데아미나아제 등의 데아미나아제를 작용시키면, 정미성이 있는 5'-뉴클레오티드류로 변환되어, 원하는 발효 조미액을 제조할 수 있다.That is, raw or heat-treated soy sauce prepared by a conventional method is desalted by electrodialysis or the like to lower the salt concentration to prepare desalted raw soy sauce or desalted heat-treated soy sauce with a salt concentration of 4% (w/v) or less. Next, the desalted raw soy sauce or desalted heat-treated soy sauce is heated in a tank with a jacket or heat-treated in a heat exchanger such as a plate heater or tube heater to inactivate phosphatase in the desalted raw soy sauce or desalted heat-treated soy sauce, and desalted raw soy sauce or Desalting heat-treated soy sauce is sterilized. Then, for sterilized desalted raw soy sauce or desalted heat-treated soy sauce, aseptically transferred to a container capable of inhibiting the incorporation of microorganisms, inoculated with nucleic acid-producing microorganisms, and nucleic acid fermentation is performed under the fermentation conditions described above. After that, the RNA extract is prepared by self-digesting the nucleic acid-producing microorganism to release the RNA in the cell body into the moromi, and a nuclease such as ribonuclease and a deaminase such as adenylate deaminase act on the RNA extract, It is converted into refined 5'-nucleotides to prepare a desired fermented seasoning solution.
본 발명의 발효 조미료는, 그대로도 천연 조미료나, 일본 농림 규격에서 정의되는 간장과 동일한 사용 방법이 가능하고, 또한 임의의 음식품에 배합할 수도 있다. 그 때문에, 본 발명의 발효 조미료를 함유한, 감칠맛(우마미)이나 깊은맛(코쿠미)이 개선된 음식품을 제공할 수 있다. 음식품의 구체예로서는, 예컨대, 간장 가공품, 쯔유, 다레, 폰즈, 일본풍 다시, 서양풍 다시, 중화 다시, 드레싱, 스프, 소스, 반찬의 원료 등의 조미료 원료; 야채, 과실, 곡물 등의 가공품을 포함하는 농산 가공 식품; 어패류, 해조 등의 가공품을 포함하는 수산 가공 식품; 계란·유제품 등의 가공품을 포함하는 축산 가공 식품; 등을 들 수 있다. 본 발명의 발효 조미료를 함유한 음식품은, 최종 제품의 핵산 함량이 높아지기 때문에, 감칠맛의 향상이나 맛있음을 높일 수 있다. 또한, 최종 제품의 원재료 표시에 있어서, 발효 조미료로서의 표시가 가능하고, 핵산의 첨가물 표시는 불필요해지는 이점을 갖는다.The fermented seasoning of the present invention can be used as a natural seasoning or the same method of use as soy sauce defined in the Japanese Agricultural Standards, and can also be blended into any food or drink. Therefore, the food-drinks containing the fermented seasoning of this invention in which umami (umami) and deep taste (kokumi) were improved can be provided. As a specific example of food-drinks, For example, seasoning raw materials, such as a soy-processed product, tsuyu, daikon, ponzu, Japanese-style dashi, Western-style dashi, Chinese dashi, dressing, soup, sauce, and side dish; Agricultural processed foods including processed products such as vegetables, fruits, and grains; Processed seafood including processed products such as seafood and seaweed; Livestock processed food including processed products such as eggs and dairy products; and the like. Since the nucleic acid content of the final product becomes high in the food-drinks containing the fermented seasoning of this invention, umami taste and taste can be improved. In addition, in the display of raw materials of the final product, it is possible to display as a fermented seasoning, and has the advantage that the display of nucleic acid additives becomes unnecessary.
이하, 실시예에 의해 본 발명을 더욱 구체적으로 설명한다. 단, 본 발명의 기술적 범위는, 이들 예에 의해 조금도 한정되는 것이 아니다.Hereinafter, the present invention will be described in more detail by way of Examples. However, the technical scope of the present invention is not limited at all by these examples.
실시예Example
(실시예 1) 고핵산 발효 조미료의 조제(Example 1) Preparation of high nucleic acid fermented seasoning
1. 간장 누룩의 제작1. Production of soy sauce yeast
탈지 가공 대두 50%(w/w)와 볶은 할쇄(割碎) 밀 50%(w/w)의 배합 비율로, 정법에 따라 간장 누룩을 제작하였다. 한편, 탈지 가공 대두는 130%(w/w) 살수하여 증자(蒸煮)한 것을 이용하였다. 이 원료에 아스페르길루스 소예(Aspergillus sojae)의 종국(種麴)을 접종하고, 25℃~40℃, 습도 95%의 환경에서 42시간 제국하여 간장 누룩을 얻었다.At a blending ratio of 50% (w/w) of defatted soybeans and 50% (w/w) of roasted whole wheat wheat, soy sauce was produced according to the recipe. On the other hand, defatted processed soybeans were steamed by spraying 130% (w/w). This raw material was inoculated with the seeds of Aspergillus sojae , and it was impregnated for 42 hours in an environment of 25 ° C. to 40 ° C. and 95% humidity to obtain soy yeast.
2. 모로미의 조제2. Preparation of Moromi
상기 간장 누룩 100 중량부에 대해, 200 중량부의 70℃로 가온한 열수(식염은 포함하지 않는다)를 혼화하고, 회전축에 교반 날개를 배치한 보온 재킷이 부착된 분해 탱크 내에서 연속적으로 100 rpm으로 교반하며, 55℃에서 24시간 가온 분해를 행하여, 무염의 모로미를 얻었다.With respect to 100 parts by weight of the soy sauce yeast, 200 parts by weight of hot water heated to 70° C. (not including salt) is mixed, and continuously at 100 rpm in a decomposition tank equipped with a thermal insulation jacket having a stirring blade disposed on the rotating shaft. While stirring, it heated and decomposed at 55 degreeC for 24 hours, and obtained the unsalted moromi.
3. 모로미의 살균 및 포스파타아제 실활 처리3. Moromi sterilization and phosphatase inactivation treatment
상기 무염 모로미 600 g을 2.5 L 용량의 자 퍼멘터(바이오트사 제조)에 넣고, 인산2수소칼륨(와코 쥰야쿠 고교사 제조) 12 g, 소포제(신에츠 실리콘사 제조) 0.6 mL, 수돗물 360 mL를 첨가하며, 수산화칼륨(와코 쥰야쿠 고교사 제조)을 첨가하여 pH 5.5가 되도록 조정하고, 오토클레이브에서 121℃, 5분의 살균을 행하였다.600 g of the unsalted moromi is placed in a 2.5 L capacity ruler Fementer (manufactured by Biot Corporation), 12 g of potassium dihydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.), 0.6 mL of an antifoaming agent (manufactured by Shin-Etsu Silicone, Inc.), and 360 mL of tap water Then, potassium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.) was added to adjust the pH to 5.5, and sterilization was performed at 121°C for 5 minutes in an autoclave.
4. 핵산 발효4. Nucleic Acid Fermentation
상기 무염 모로미를 30℃로 냉각하고, 핵산 생산 효모로서 칸디다 유틸리스(Candida utilis)를 식균하며, 정법에 따라 30℃에서 48시간의 배양을 행하였다. 별도로 멸균한 50% 글루코오스 수용액을 1.7 g/hr로 유가(流加)하고, 무균 공기를 1 L/min, 내압 0.04 ㎫로 통기하였다. 교반 속도는 600 rpm으로 하였다.The unsalted Moromi was cooled to 30° C., and Candida utilis was inoculated as a nucleic acid producing yeast, followed by culturing at 30° C. for 48 hours according to the conventional method. A separately sterilized 50% aqueous glucose solution was fed at 1.7 g/hr, and sterile air was vented at 1 L/min and an internal pressure of 0.04 MPa. The stirring speed was 600 rpm.
5. 효소 처리5. Enzyme Treatment
배양 종료 후, 투니카제(상표, 아마노 엔자임사 제조)를 300 ㎎ 첨가하고, 50℃에서 2시간 교반하였다. 계속해서, 뉴클레아제 아마노 G(상표, 아마노 엔자임사 제조)를 300 ㎎ 첨가하고, 65℃에서 1시간 교반하였다. 또한, 데아미자임(상표, 아마노 엔자임사 제조)을 300 ㎎ 첨가하고, 45℃에서 1시간 교반하였다. 마지막으로 80℃에서 20분간 교반하여 효소를 실활시키고, 정법에 따라 여과지(어드밴텍사 제조)로 여과를 행하여, 핵산을 고함유하는 발효(간장풍) 조미액을 얻었다(샘플 1).After completion of the culture, 300 mg of tunicase (trademark, manufactured by Amano Enzyme) was added, and the mixture was stirred at 50°C for 2 hours. Then, 300 mg of nuclease Amano G (trademark, manufactured by Amano Enzyme) was added, and the mixture was stirred at 65°C for 1 hour. Furthermore, 300 mg of Deamizyme (trademark, manufactured by Amano Enzyme) was added, and the mixture was stirred at 45°C for 1 hour. Finally, the enzyme was inactivated by stirring at 80° C. for 20 minutes, followed by filtration with filter paper (manufactured by Advantech) according to the usual method to obtain a fermented (soy sauce-style) seasoning solution containing a high nucleic acid (Sample 1).
6. 발효 조미료의 성분 분석6. Component Analysis of Fermented Seasonings
(1) 정미성 핵산의 정량(1) Quantitation of unqualified nucleic acids
정미성 핵산의 정량은, 기보(旣報)(동경 도립 위생 연구소 연구 연보, p172-175, 2001년)를 개변한 방법으로, 고속 액체 크로마토그래피(HPLC)를 이용하여 행하였다. 즉, 고상 추출 칼럼 Sep-Pak Light QMA(워터스사 제조)를 이용하여, 하기의 요령으로 전처리를 행하였다.The quantitative nucleic acid was quantified using high performance liquid chromatography (HPLC) by the method modified|transformed by Kibo (Research Yearbook of Tokyo Metropolitan Sanitary Research Institute, p172-175, 2001). That is, using the solid phase extraction column Sep-Pak Light QMA (manufactured by Waters), pretreatment was performed in the following manner.
↓메탄올 2 mL(활성화)↓Methanol 2mL (activated)
↓초순수 4 mL(평형화)↓ 4 mL of ultrapure water (equilibration)
↓샘플 300 uL↓Sample 300 uL
(암모니아 50 uL와 혼합하여 350 uL를 로드)(Load 350 uL mixed with 50 uL of ammonia)
↓초순수 3 mL(비흡착 분획)↓ 3 mL of ultrapure water (non-adsorbed fraction)
↓1% 포름산 5 mL(흡착 분획)↓ 5 mL of 1% formic acid (adsorption fraction)
↓원심 농축 장치로 증발 건고(乾固)↓ Evaporation to dryness by centrifugal concentrator
↓HPLC의 용리액 A에 재용해↓Redissolve in Eluent A of HPLC
다음으로, 이 전처리가 완료된 샘플을 HPLC(시마즈 세이사쿠쇼사 제조)로 분석하였다. 조건은 하기와 같다.Next, the sample on which this pretreatment was completed was analyzed by HPLC (manufactured by Shimadzu Corporation). The conditions are as follows.
칼럼: Shiseido CAPCELL PAK C18 MGIII 4.6x250 ㎜(시세이도사 제조)Column: Shiseido CAPCELL PAK C18 MGIII 4.6x250 mm (manufactured by Shiseido)
용리액 A: 0.1% 트리에틸아민+1% 메탄올/초순수, pH 5.0(인산으로 조정)Eluent A: 0.1% triethylamine + 1% methanol / ultrapure water, pH 5.0 (adjusted with phosphoric acid)
용리액 B: 80% 아세토니트릴/초순수Eluent B: 80% acetonitrile/ultra-pure water
유속: 1.0 mL/min, 검출: UV 254 ㎚Flow rate: 1.0 mL/min, detection: UV 254 nm
표준품으로서 이용한 5'-IMP 및 5'-GMP의 검량선으로부터, 정량을 행하였다. 그 결과, 핵산 농도는 표 1과 같았다. 단, 농도는 5. 효소 처리에서 얻은 발효 조미액의 액량당 농도(w/v)이다.Quantification was performed from the calibration curves of 5'-IMP and 5'-GMP used as standards. As a result, the nucleic acid concentration was shown in Table 1. However, the concentration is the concentration (w/v) per liquid volume of the fermentation seasoning obtained in 5. Enzyme treatment.
(2) HDMF의 정량(2) Quantification of HDMF
HDMF의 농도는, J.Agric.Food Chem.Vol.39, 934, 1991에 기재된 정량 분석법에 의해 실시하였다. 보다 구체적으로는, 가스 크로마토그래피(애질런트·테크놀로지스사 제조 6890N)에 의한 분석을 행하고, 표준 물질을 이용한 검량선법에 의해, 각종 향기 성분 함유량을 결정하였다.The density|concentration of HDMF was implemented by the quantitative analysis method described in J.Agric.Food Chem.Vol.39, 934, 1991. More specifically, analysis by gas chromatography (6890N by Agilent Technologies) was performed, and various fragrance component content was determined by the analytical curve method using the standard substance.
(실시예 2) 간장풍 조미액에 핵산을 첨가한 효과(Example 2) Effect of adding nucleic acids to soy sauce-style seasoning
일본 특허 제5836466호 공보의 실시예 2-1에 기재된 방법으로 조제한 무염 간장풍 조미료에, 식염 농도가 4%(w/v) 미만이 되도록 식염을 첨가하고, 또한 5'-IMP 및 5'-GMP를 주성분으로서 함유하는 조미료(상품명: 리보타이드, MC 푸드 스페셜리티스사 제조, 5'-IMP:5'-GMP 함유량비=1:1)를 각 농도로 첨가하여, 원하는 간장풍 조미액을 조제하였다(샘플 2~7). 이들 각 샘플에 대해, 이하의 요령으로 관능 평가를 행하였다. 한편, 대조의 감염 간장에 원래 포함되는 핵산량은 검출 한계 이하인 것을 분석에 의해 확인하였다. 관능 평가는, 식별 능력을 갖는 패널 7명이 샘플의 원액을 스포이드로 직접 입에 머금고, 핵산을 첨가하고 있지 않은 대조의 간장풍 조미액과 맛에 차이가 있는지의 여부를 평가하여, 차이를 식별할 수 있었던 사람 수로 나타내었다. 또한, 패널 전원의 토의에 의해, 종합적인 맛의 바람직함을 기호로 나타내었다(표 2). 한편, 기호는 이하의 평가를 나타낸다. ◎: 대조와 비교하여 매우 바람직하다, ○: 대조와 비교하여 바람직하다, ×: 대조와 동등.To the unsalted soy sauce-style seasoning prepared by the method described in Example 2-1 of Japanese Patent No. 5836466, salt was added so that the salt concentration was less than 4% (w/v), and 5'-IMP and 5'- A seasoning containing GMP as a main ingredient (trade name: Ribotide, manufactured by MC Food Specialties, 5'-IMP:5'-GMP content ratio = 1:1) was added at each concentration to prepare a desired soy sauce-style seasoning ( samples 2-7). For each of these samples, sensory evaluation was performed in the following manner. On the other hand, it was confirmed by analysis that the amount of nucleic acid originally contained in the control infected liver was below the detection limit. For sensory evaluation, 7 panelists with discernment ability put the sample stock solution directly in their mouth with a dropper, and evaluate whether there is a difference in taste from the soy sauce-style seasoning solution of the control without nucleic acid added to identify the difference. expressed as the number of people who could. In addition, according to the discussion of all the panelists, the desirability of the overall taste was indicated by preference (Table 2). In addition, a symbol shows the following evaluation. (double-circle): very preferable compared with control, (circle): compared with control, x: equivalent to control.
표 2로부터, 핵산 농도가 10 ppm 이상인 샘플(샘플 3~7)에서는 패널 전원이 맛의 차이를 식별하고, 특히 100 ppm 이상에서는 감칠맛이나 맛의 지속성이 향상되어 있다고 하는 코멘트가 있었다. 이 결과로부터, 본 발명에 유사한 제조법에 의해 얻어지는 간장풍 조미료에, 10 ppm 이상의 핵산을 함유시킴으로써, 간장풍 조미료의 맛을 개선할 수 있는 것으로 나타났다. 단, 핵산 농도가 10000 ppm에서는, 핵산의 맛이 지나치게 강하여, 간장풍 조미료로서의 맛의 밸런스가 무너진다고 하는 패널의 코멘트가 있었기 때문에, 종합 평가로서는 대조와 동일한 정도가 되었다.From Table 2, in the samples (
(실시예 3) 간장에 핵산 함유 발효 조미료를 첨가한 효과(Example 3) Effect of adding nucleic acid-containing fermented seasoning to soy sauce
감염 간장(기꼬만사 제조, 시판품)에, 본 발명의 실시예 1에서 조제한 핵산 함유 간장풍 조미료(샘플 1)를 배합량을 변경해서 첨가하여, 핵산 농도가 1~1000 ppm인 간장풍 조미액을 조제하였다. 이들 각 샘플에 대해, 이하의 요령으로 식염 농도가 9%(w/v)가 되도록 식염을 첨가하여, 원하는 간장풍 조미료(샘플 8~12)를 조제하였다. 이들 각 샘플에 대해, 실시예 2와 동일한 방법으로 관능 평가를 행하였다.To infected soy sauce (manufactured by Kikkoman, commercially available product), the nucleic acid-containing soy sauce-style seasoning (Sample 1) prepared in Example 1 of the present invention was added in varying amounts to prepare a soy sauce-style seasoning having a nucleic acid concentration of 1-1000 ppm. . To each of these samples, salt was added so that the salt concentration became 9% (w/v) in the following manner, and desired soy sauce-style seasonings (Samples 8 to 12) were prepared. For each of these samples, sensory evaluation was performed in the same manner as in Example 2.
표 3으로부터, 핵산 농도가 10 ppm 이상이 되도록 본 발명의 핵산 함유 발효 조미료를 첨가하면, 감염 간장의 맛이 현저히 개선되는 것이 확인되었다. 한편, 핵산 농도가 1000 ppm에서는, 핵산 함유 발효 조미료의 배합량이 비교적 많았기 때문에, 대조의 감염 간장과는 약간 다른 맛이 느껴졌으나, 전체로서는 품질이 개선되었다. 또한, 본 발명에서는 현저한 양의 HDMF를 함유하기 때문에, 간장과 같은 향기로운 향기를 갖고, 발효 조미료로서 우수한 품질을 갖는 것이 확인되었다.From Table 3, it was confirmed that the taste of the infected soy sauce was significantly improved when the nucleic acid-containing fermented seasoning of the present invention was added so that the nucleic acid concentration was 10 ppm or more. On the other hand, at a nucleic acid concentration of 1000 ppm, since the amount of the fermented seasoning containing nucleic acid was relatively large, the taste was slightly different from that of the control infected soy sauce, but the overall quality was improved. Further, in the present invention, it was confirmed that since it contained a remarkable amount of HDMF, it had a fragrant aroma like soy sauce and had excellent quality as a fermented seasoning.
(실시예 4) 핵산 분해 효소의 실활 조건(Example 4) Conditions for inactivation of nucleolytic enzymes
실시예 1의 무염 모로미 4 g에 수돗물 6 mL를 첨가하고, 이것을 50, 60, 70, 80℃에서 10분간, 또는 121℃에서 2분간의 조건으로 가열하였다. 계속해서, 각 모로미에 5'-구아닐산 900 ppm, 5'-이노신산 900 ppm을 첨가하고, 30℃에서 46시간 진탕하였다. 그 후, 원심 분리에 의해 고형분을 제거하고, 상청액을 0.45 ㎛ 필터(어드밴텍사 제조)에 통과시켰다.6 mL of tap water was added to 4 g of unsalted moromi of Example 1, and this was heated at 50, 60, 70, 80°C for 10 minutes, or at 121°C for 2 minutes. Then, 900 ppm of 5'-guanylic acid and 900 ppm of 5'-inosinic acid were added to each moromi, and the mixture was shaken at 30°C for 46 hours. Thereafter, the solid content was removed by centrifugation, and the supernatant was passed through a 0.45 µm filter (manufactured by Advantech).
정미성 핵산의 정량은 고속 액체 크로마토그래프 질량 분석계(LC-MS)를 이용하여 행하였다. 조건은 하기와 같다.The quantitative nucleic acid was quantified using a high performance liquid chromatograph mass spectrometer (LC-MS). The conditions are as follows.
칼럼: Poroshell 120 PFP 3.0x150 ㎜(애질런트사 제조)Column: Poroshell 120 PFP 3.0x150 mm (manufactured by Agilent)
용리액 A: 0.1% 포름산/초순수Eluent A: 0.1% formic acid/ultra-pure water
용리액 B: 0.1% 포름산/아세토니트릴Eluent B: 0.1% formic acid/acetonitrile
유속: 0.4 mL/minFlow rate: 0.4 mL/min
이온화: ESI(+)Ionization: ESI(+)
검출: SIM 모드Detection: SIM mode
표준 첨가법에 따라, 5'-IMP 및 5'-GMP의 표품(標品)을 이용하여 정량을 행하였다. 그 결과를 도 1에 도시한다. 도 1은 무염 모로미를 가열한 후에 정미성 핵산(5'-구아닐산 및 5'-이노신산)을 첨가하고, 경시적으로 핵산 농도를 정량한 결과를 도시한 도면이다.According to the standard addition method, quantification was performed using 5'-IMP and 5'-GMP samples. The results are shown in FIG. 1 . 1 is a view showing the results of quantifying the concentration of nucleic acids over time after heating unsalted moromi, and then adding crude nucleic acids (5'-guanylic acid and 5'-inosine acid).
도 1에 도시된 바와 같이, 가열 온도가 70℃ 이하인 경우에는 18시간 처리한 시점에서 정미성 핵산이 완전히 분해되었으나, 가열 온도 80℃ 이상인 경우에는 처리 개시로부터 46시간까지 정미성 핵산은 분해되지 않았다. 따라서, 핵산 분해 효소를 실활시키기 위해서는 80℃ 10분간의 멸균이 필요해지는 것이 판명되었다.As shown in FIG. 1 , when the heating temperature was 70° C. or lower, the crude nucleic acid was completely decomposed at the time of treatment for 18 hours, but when the heating temperature was 80° C. or higher, the crude nucleic acid was not decomposed from the start of the treatment to 46 hours. . Therefore, it became clear that sterilization for 10 minutes at 80 degreeC was required in order to inactivate a nuclease.
(실시예 5) 각종 효모에 의한 핵산 발효(Example 5) Nucleic acid fermentation by various yeasts
실시예 1의 무염 모로미 40 g에, 인산2수소칼륨 1 g, 소포제 0.1 g, 수돗물 60 mL를 첨가하고, 수산화칼륨으로 pH 5.5가 되도록 조정하며, 오토클레이브에서 121℃, 2분의 살균을 행하였다.To 40 g of unsalted moromi of Example 1, 1 g of potassium dihydrogen phosphate, 0.1 g of an antifoaming agent, and 60 mL of tap water were added, adjusted to pH 5.5 with potassium hydroxide, and sterilized at 121° C. for 2 minutes in an autoclave. did
상기 배지에 별도로 멸균한 50% 글루코오스 용액을 5 mL 첨가하고, 칸디다 유틸리스(Candida utilis)(샘플 13) 또는 사카로마이세스 세레비시에(Saccharomyces cerevisiae)(샘플 14) 또는 자이고사카로마이세스 룩시이(Zygosaccharomyces rouxii)(샘플 15) 또는 클루이베로마이세스 마르시아누스(Kluyveromyces marxianus)(샘플 16)를 식균하며, 정법에 따라 30℃에서 24시간의 배양을 행하였다.5 mL of a separately sterilized 50% glucose solution was added to the medium, and Candida utilis (Sample 13) or Saccharomyces cerevisiae (Sample 14) or Zygosaccharomyces rook Shii ( Zygosaccharomyces rouxii ) (Sample 15) or Kluyveromyces marxianus (Sample 16) was inoculated, and cultured at 30° C. for 24 hours according to the conventional method.
배양 종료 후, 배양액 20 mL로부터 원심 분리로 상청을 제거하고, 얻어진 침전을 물에 현탁하여 효모 현탁액 20 mL를 조제하였다. 이 효모 현탁액에 에탄올 0.5 mL를 첨가한 후, pH를 8로 조정하고, 50℃에서 2시간 진탕하였다. 계속해서, pH를 5로 조정하고, 뉴클레아제 아마노 G(상표, 아마노 엔자임사 제조)를 7.5 ㎎ 첨가하며, 65℃에서 1시간 반응시켰다. 또한, 데아미자임(상표, 아마노 엔자임사 제조)을 7.5 ㎎ 첨가하고, 55℃에서 1시간 반응시켰다. 마지막으로 85℃에서 20분간 가온하여 효소를 실활시키고, 원심 분리에 의해 고형분을 제거하여, 핵산 발효 조미액(샘플 13~16)을 얻었다.After completion of the culture, the supernatant was removed from 20 mL of the culture solution by centrifugation, and the resulting precipitate was suspended in water to prepare 20 mL of a yeast suspension. After adding 0.5 mL of ethanol to this yeast suspension, pH was adjusted to 8, and it shook at 50 degreeC for 2 hours. Then, the pH was adjusted to 5, 7.5 mg of nuclease Amano G (trademark, manufactured by Amano Enzyme) was added, and the reaction was carried out at 65°C for 1 hour. Furthermore, 7.5 mg of Deamizyme (trademark, manufactured by Amano Enzyme) was added, and it was made to react at 55 degreeC for 1 hour. Finally, the enzyme was inactivated by heating at 85° C. for 20 minutes, and the solid content was removed by centrifugation to obtain a nucleic acid fermentation seasoning (Samples 13 to 16).
실시예 4와 동일한 조건으로 핵산 발효 조미액을 분석한 결과, 핵산 농도는 표 4와 같았다. 표 4로부터, 칸디다 유틸리스 이외의 효모도 핵산 발효 조미액의 제조에 사용할 수 있는 것으로 나타났다.As a result of analyzing the nucleic acid fermentation seasoning under the same conditions as in Example 4, the nucleic acid concentrations were as shown in Table 4. From Table 4, it was shown that yeast other than Candida utilis can also be used for the preparation of the nucleic acid fermentation broth.
(실시예 6) HDMF 농도의 비교(Example 6) Comparison of HDMF concentration
본 발명품과 시판의 고핵산 효모 엑기스에 포함되는 HDMF 농도를 비교하기 위해서, 핵산 발효 조미액을 제작하였다.In order to compare the HDMF concentration contained in the product of the present invention and a commercially available high nucleic acid yeast extract, a nucleic acid fermentation seasoning solution was prepared.
실시예 1의 무염 모로미 520 g에, 인산2수소칼륨 13 g, 소포제 0.6 g, 수돗물 380 mL를 첨가하고, 수산화칼륨으로 pH 5.5가 되도록 조정하며, 오토클레이브에서 115℃, 15분의 살균을 행하였다. 이 배지에 칸디다 유틸리스를 식균하고, 정법에 따라 30℃에서 22시간의 배양을 행하였다. 별도로 멸균한 50% 글루코오스 수용액을 8.4 g/hr로 유가하고, 무균 공기를 1 L/min, 내압 0.04 ㎫로 통기하였다. 교반 속도는 600 rpm으로 하였다.To 520 g of unsalted moromi of Example 1, 13 g of potassium dihydrogen phosphate, 0.6 g of an antifoaming agent, and 380 mL of tap water were added, adjusted to pH 5.5 with potassium hydroxide, and sterilized at 115° C. for 15 minutes in an autoclave. did Candida utilis was inoculated into this medium, and cultured at 30 DEG C for 22 hours according to the conventional method. A separately sterilized 50% aqueous glucose solution was added at a rate of 8.4 g/hr, and sterile air was vented at 1 L/min and an internal pressure of 0.04 MPa. The stirring speed was 600 rpm.
배양 종료 후, 배양액 30 mL에 에탄올 1 mL를 첨가한 후, pH를 6.5로 조정하고, 50℃에서 2시간 진탕하였다. 계속해서, pH를 5로 조정하고, 뉴클레아제 아마노 G(아마노 엔자임사 제조)를 15 ㎎ 첨가하며, 70℃에서 1시간 반응시켰다. 반응 후, pH를 6.5로 조정하고, 데아미자임(아마노 엔자임사 제조)을 15 ㎎ 첨가하며, 55℃에서 1시간 반응시켰다. 마지막으로 85℃에서 20분간 가온하여 효소를 실활시키고, 원심 분리에 의해 고형분을 제거하여, 핵산 발효 조미액(샘플 17)을 얻었다.After completion of the culture, 1 mL of ethanol was added to 30 mL of the culture solution, the pH was adjusted to 6.5, and the mixture was shaken at 50°C for 2 hours. Then, the pH was adjusted to 5, 15 mg of nuclease Amano G (manufactured by Amano Enzyme) was added, and the reaction was carried out at 70°C for 1 hour. After the reaction, the pH was adjusted to 6.5, 15 mg of deazyme (manufactured by Amano Enzyme) was added, and the reaction was carried out at 55°C for 1 hour. Finally, the enzyme was inactivated by heating at 85°C for 20 minutes, and the solid content was removed by centrifugation to obtain a nucleic acid fermentation seasoning (Sample 17).
아로마일드(상표, 고진 라이프 사이언스사 제조) 0.25 g을 50 mL의 정수에 첨가하고, 이것을 0.1% 포름산 함유수로 50배 희석한 것을 정미성 핵산의 분석에 이용하였다. HDMF의 정량에는 아로마일드 50 ㎎을 50 mL의 정수에 첨가한 것을 이용하였다. HDMF의 정량과 정미성 핵산의 정량 조건은 실시예 1에 기재한 조건과 같다. 0.25 g of Aromaild (trademark, manufactured by Gojin Life Sciences) was added to 50 mL of purified water, and this was diluted 50-fold with 0.1% formic acid-containing water, which was used for analysis of crude nucleic acids. For quantification of HDMF, 50 mg of Aromaylde added to 50 mL of purified water was used. The conditions for quantitation of HDMF and quantification of clean nucleic acids are the same as those described in Example 1.
결과를 표 5에 나타낸다. 표 5에 기재되어 있는 바와 같이, 시판 진간장은 정미성 핵산이 검출 한계 이하였으나, 본 발명의 발효 조미액(샘플 17)은 정미성 핵산을 현저한 양 함유하고 있었다. 또한, 본 발명의 발효 조미액(샘플 17)은 시판 진간장과 동등한 HDMF를 함유하고 있었으나, 시판 고핵산 효모 엑기스로부터는 HDMF가 검출되지 않았다.A result is shown in Table 5. As shown in Table 5, the commercial dark soy sauce had a level of nucleic acid below the detection limit, but the fermented seasoning broth (Sample 17) of the present invention contained a significant amount of the refined nucleic acid. In addition, the fermented seasoning liquid (Sample 17) of the present invention contained HDMF equivalent to that of commercially available soy sauce, but HDMF was not detected from the commercially available high nucleic acid yeast extract.
따라서, 본 발효 조미액은 무첨가로 핵산을 고함유하고, 간장과 같은 향기로운 풍미가 우수한 조미액으로서 제공할 수 있는 것이 판명되었다.Therefore, it has been found that the present fermented seasoning solution can be provided as a seasoning solution having a high content of nucleic acids without additives and excellent in fragrant flavor like soy sauce.
Claims (10)
4-히드록시-2,5-디메틸-3(2H)-퓨라논(HDMF) 2 ppm(w/v) 이상
을 함유하는
식염 농도가 4%(w/v) 이하인 모로미를 이용하여 얻어진 발효 조미료.50 ppm (w/v) or more of nucleic acids (excluding nucleic acids derived from addition);
4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) 2 ppm (w/v) or more
containing
A fermented seasoning obtained by using Moromi with a salt concentration of 4% (w/v) or less.
상기 모로미를 가열 처리하여 포스파타아제를 실활시킴으로써 살균 모로미를 조제하는 공정과,
상기 살균 모로미에 핵산 생산 미생물을 접종하여 위해 미생물의 혼입을 억제할 수 있는 용기에서 핵산 발효를 행하는 공정과,
상기 핵산 생산 미생물을 자기 소화하여 균체 내의 RNA를 모로미 중에 유리시킴으로써 RNA 추출물을 조제하는 공정과,
상기 RNA 추출물에 뉴클레아제를 작용시켜 정미성이 있는 5'-뉴클레오티드류로 변환하는 공정
을 갖는
핵산(단, 첨가 유래의 핵산을 제외한다) 50 ppm(w/v) 이상을 함유하는 발효 조미료의 제조 방법.A step of heating and decomposing moromi with a salt concentration of 4% (w/v) or less by adding water or saline to a grain raw material containing soybeans or wheat as the main raw material, preparing solid yeast by inoculating koji bacteria;
a step of preparing sterilized moromi by heat-treating the moromi to inactivate phosphatase;
A step of inoculating the sterilization moromi with a nucleic acid-producing microorganism and performing nucleic acid fermentation in a container capable of suppressing the incorporation of harmful microorganisms;
A step of preparing an RNA extract by self-digesting the nucleic acid-producing microorganism to release the RNA in the microbial cells in Moromi;
A process of converting the RNA extract into refined 5'-nucleotides by acting with a nuclease
having
A method for producing a fermented seasoning comprising 50 ppm (w/v) or more of nucleic acids (excluding nucleic acids derived from addition).
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| CN110301612A (en) * | 2019-07-25 | 2019-10-08 | 上海太太乐食品有限公司 | Utilize corn fermentation sauce/powder preparation solid condiment and granulation/flouring technology |
| CN111748592B (en) * | 2020-07-17 | 2022-03-15 | 通辽梅花生物科技有限公司 | Preparation method of high-purity flavor nucleotide disodium |
| CN112772896A (en) * | 2021-03-09 | 2021-05-11 | 仇俊鹏 | Soybean and wheat-derived soy sauce with natural meat flavor and its preparation method |
| CN113100429A (en) * | 2021-05-27 | 2021-07-13 | 山东玉兔食品股份有限公司 | Process for producing high-nucleotide super-fresh soy sauce |
| CN114480475B (en) * | 2021-12-29 | 2023-05-30 | 中国科学院海洋研究所 | Recombinant Porphyra yezoensis strain rich in flavor substances, construction method and application |
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| JP2006525794A (en) | 2003-01-27 | 2006-11-16 | デーエスエム アイピー アセッツ ベー． ヴェー． | Production of 5'-ribonucleotides |
| JP4781365B2 (en) * | 2005-09-30 | 2011-09-28 | キッコーマン株式会社 | Soy sauce containing 5'-nucleotides and method for producing the same |
| JP2016059382A (en) | 2014-09-12 | 2016-04-25 | キッコーマン株式会社 | Manufacturing method of soy sauce-like seasoning and soy sauce-like seasoning |
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